Iron (Fe) deficiency is a nutritional disorder that affects nearly two billion people worldwide. The long-term goal of the proposed research is to understand how organisms cope with Fe-deficiency using the yeast Saccharomyces cerevisiae as a model system. The specific hypothesis is that the mRNA-binding proteins Cth1 and Cth2 cooperatively mediate the adaptation to Fe-limiting conditions in S. cerevisiae through promoting targeted mRNA decay. This hypothesis is based on the observations that 1) during Fe-deficiency, Cth2 promotes the down regulation of many mRNAs involved in metabolic pathways, and cth2 mutant cells grow poorly on medium lacking Fe;2) deletion of the CTH1 gene on a cth2 strain further exacerbates the growth phenotype on low Fe conditions and cth1cth2 mutants fail to down regulate several of the mRNAs targeted for turnover during Fe-deficiency;and 3) concomitantly with the down regulation of many mRNAs, wild type cells show increased steady-state levels of glucose-regulated genes under low Fe, but not cth1cth2 cells under the same conditions. The specific aims are to: 1. Establish the contribution of Cth1 protein to the response to Fe limitation. The transcriptional activation, mRNA-binding and degradation-promoting activities of Cth1 in response to Fe-deficiency will be ascertained using traditional techniques such Chromatin ImmunoPrecipitation (ChIP), beta-galactosidase reporter assays, yeast three-hybrid assays and RNA blotting. Microarray analyses will be performed to identify specific targets of Cth1 and Cth2 in response to low Fe. 2. Establish the effects of Cth1 and Cth2 activity on glucose metabolism during Fe-deficiency. Preliminary data suggest that Fe-starved yeast express several glucose-repressed genes, but fail to do so in the absence of Cth1 and Cth2. Glucose utilization during Fe-deficiency will be monitored using enzymatic assays as well as examining phospho-Snfl and fructose-1, 6-bisphosphatase levels in wild type cells and in cth1cth2 mutants. In addition, non-mRNA binding alleles of Cth1 and Cth2 will be used to ascertain whether their decay-promoting activity is necessary for the secondary glucose starvation response. Iron (Fe) deficiency is the number one nutritional disorder affecting nearly two billion people worldwide. The goal of this research is to understand how eukaryotes adjust their metabolism to conditions of Fe-deficiency using the genetically tractable yeast Saccharomyces cerevisiae, which will contribute to a better understanding of how humans cope with cellular Fe-deficiency at the molecular level.